Carex borealifujianica (Cyperaceae), a new species of the core Carex clade from Fujian, southeastern China

A new species, Carex borealifujianica Y.F. Lu & X.F. Jin (Cyperaceae, sect. Occlusae of core Carex clade) is described and illustrated from northern Fujian, China. In addition to morphological comparisons with its relatives, comparative micromorphology of utricles and achenes of seven species in Carex sect. Occlusae was examined. Micromorphology of utricles and achenes revealed the similarity of Carex borealifujianica and C. ligulata. Morphologically, this new species is similar to Carex ligulata in having lateral spikes remote and densely flowered, as well as utricles densely hispidulous, but differs in having 2 or 3 narrowly clavate staminate spikes, leaves 2.5–5 mm wide with sheaths sparsely pilose, and achenes emarginate at the apex. The phylogenetic analysis from two nuclear DNA regions (ETS and ITS) and two chloroplast DNA regions (matK and trnL-F) of 68 taxa resolved C. borealifujianica as a distinct species.


Introduction
Carex L., a cosmopolitan genus containing ca. 2000 known species divided into 130+ sections, is one of the largest genera of flowering plants [1][2][3][4]. The genus is distributed across every continent with the exception of Antarctica and grows in various habitats ranging from tropical forests to Arctic tundra [5]. The genus Carex is unique within the family Cyperaceae and characterized by its unisexual flowers with pistillate flowers surrounded by a fully or partially connate sac-like structure (utricle) [6].
The genus Carex (sedges) is also notoriously difficult in both taxonomy and phylogeny. In the past ten years, phylogenetic studies revealed that Carex as traditionally circumscribed is largely paraphyletic [6,7]. The genus Carex now includes Cymophyllus Mack., Kobresia Willd., Schoenoxiphium Nees, and Uncinia Pers. Recent studies revealed Carex is comprised of six major clades corresponding to six subgenera: 1) the Siderosticta clade (subg. Siderosticta Waterway), sister to the rest of the genus and the earliest diverging lineage, with large chromosomes but low numbers of chromosomes; 2) the Schoenoxiphium clade (

Nomenclature
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Population sampling and specimen examination
During our explorations on Mount Wuyi in 2018 and 2019, we collected a Carex with densely hispidulous utricles. The plant was easily recognized as a member of Carex sect. Occlusae C.B. Clarke.
A total of ten populations were examined for morphological analysis, including one population of the new species (Carex borealifujianica), six populations of C. ligulata, and three populations of C. hebecarpa. The specimens are conserved in the following herbaria: Hangzhou Normal College (HTC), South China Botanical Garden (IBSC), Institute of Botany, Chinese Academy of Sciences (PE) and University of Tokyo (TI), and collection information is shown in Table 2.

Morphological observations and statistical analysis
Morphological terminology used in the description of the species mainly follows Dai et al.  [11,14]. Quantitative characters, including culm height, leaf width, number, length and width of staminate and pistillate spikes, length of peduncle of staminate spike, pistillate flowers per spike, length of pistillate scales, utricle and achenes, were measured on all the samples. The density of utricles was calculated by utricle number per spike length, and the other qualitative characters were directly obtained and assigned by examining specimens. The size measurements of utricles and achenes of each individual were randomly measured from a total of 20 utricles and achenes of different spikes. Eighty individuals and 17 diagnostic characters were analyzed, and all diagnostic characters are presented in Table 3. A data matrix using 17 characters was produced for each population with each individual as an operational taxonomic unit (OTU) ( Table 3). SPSS version 11.5 software was used to analyze the variation of quantitative characters, and principal component analysis (PCA) was used to determine the similarities among populations based on 17 characters in RStudio [40] using the functions of "scale", "princomp" and "predict"; a 95% confidence interval was employed using the function "stat_ellipse" in the "ggplot2" package.

SEM observations
Scanning electron microscope (SEM) observations were conducted to compare the detailed micromorphology of utricles and achenes. The utricles and achenes of Carex borealifujianica and the other six species of Carex sect. Occlusae examined were studied from specimens. Mature utricles were cleaned in 50% ethanol for 2 hr, and air dried. The cleaned utricles were mounted on stubs using double-sided adhesive tape, and coated with a layer of gold [22]. Mature achenes were initially soaked in a solution of concentrated sulfuric acid and acetic anhydride (vol.: vol. = 1: 9) for 12-18 hr, then rinsed in acetic acid for 10 min and water for 5 min, respectively. These samples were placed in a bath-type ultrasonic cleaner for 30 min with 70% ethanol to remove the cuticle and outer periclinal wall of the epidermis [41]. After air drying, the achenes were mounted and coated in a gold layer. The coated utricles and achenes were observed and photographed using an XL-30E scanning electron microscope (Philips, The Netherlands).

Taxon sampling, DNA extraction, PCR and sequencing
A total of 22 samples representing 18 taxa were used for molecular phylogenetic analyses based on the results of the recent global megaphylogeny of Carex [11,12], which indicated sect.
Occlusae was a member of core Carex clade and closely related to sects. Hallerianae, Acrocystis and Clandestinae; 48 sequences were newly generated in this study (including those of 2 species, C. phyllocephala and C. maubertiana, which were sequenced for the first time); the other sequences were directly downloaded from GenBank (see Table 1).
Total genomic DNA was extracted from leaves dried in silica gel using TIANGEN Plant Genomic DNA Kit (Tiangen Biotech, Beijing, China). Two nuclear DNA regions (ETS and ITS) and two chloroplast DNA regions (matK and trnL-F) used for phylogenetic analysis, were amplified using a DNA Engine PCR machine (Bio-Rad, Hercules, CA, USA) in 25 μL reactions. The amplification reaction mixture was prepared using Golden DNA Polymerase following the manufacturer's instructions (Tiangen Biotech) and the amplified primers followed Starr et al. [42] for ETS, White et al. [43] for ITS, Starr and Ford [44] for matK and Taberlet et al. [45] for trnL-F. The PCR program began at 94˚C for 4 min, followed by 37 cycles of 94˚C for 30 s, 55˚C (57˚C for ITS) for 30 s and 72˚C for 1 min, followed by a 72˚C extension for 10 min. All PCR products were electrophoresed on 1% agarose gels to verify product size.
The PCR products were purified with an AxyPrep PCR Clean-up Kit (Axygen Biotechnology Co., Hangzhou, China) following the manufacturer's instructions. Sequencing was carried out on an ABI 3730 automated sequencer (Applied Biosystems, Foster City, CA, USA).

Molecular phylogenetic analysis
Sequences were aligned using the online version of MAFFT [46], and then manually adjusted by MEGA X [47]. The ETS, ITS, trnL-F and matK were combined in SequenceMatrix [48]. Optimal DNA substitute models for each region were selected using Akaike information criterion [49] in jModeltest v.2.1.3 [50], and resulted in GTR+G for ETS, ITS and trnL-F, GTR +I+G for matK. Phylogenetic analysis was conducted in RAxML v. 8 [51] for Maximum Likelihood (ML) and Mrbayes v. 3.2.6 for Bayesian Inference (BI) [52]. For the ML analysis, a bootstrap analysis with 1000 replicates was conducted with a GTR+G model using other parameters by default. For the Bayesian analysis, four Metropolis-coupled Markov-chains Monte Carlo were run for 10 7 generations with sampling every 1000 generations and until the standard deviation of split frequencies fell below 0.01. The first 2500 recovered trees were discarded (burn in = 0.25). Maximum parsimony bootstrap (BP) and Bayesian posterior probability (PP) were respectively calculated.

Assessment of conservation status
International Union for Conservation of Nature (IUCN) Red List Categories and Criteria in 2012 was used to assess the conservation status of the new species. We followed the definitions and categories and used the field investigation data to estimate, such as population size, geographic range, and quality of habitat.

Morphological variation within and among populations
The morphological characters we observed and measured are shown in Table 3 (see S1 Data), which indicates that most of the quantitative characters in the analysis are variable both within and across populations. In the PCA analysis, four principal components with eigenvalues >1 were extracted; these explained 39.61%, 19.78%, 11.85% and 6.09% of the variation among individuals, respectively. The first principal component had the highest loadings for leaf sheath indumentum, whether or not utricles had distinct thin veins, leaf width, indumentum of pistillate scales, number of staminate spikes, and density of utricles. The second principal component had the highest loadings for pistillate scale, utricle, and pistillate spike lengths. Based on the PCA of the diagnostic morphological characters, three groups were clearly recognized. The population named FJ-B was isolated from the other nine populations, whereas three populations (SC-H, TI-H and YN-H) formed the second group, close to the third group which included five populations from China (AH-L, FJ-L, HN-L, JX-L, and SCCQ-L) and one from Japan (JA-L). These three groups were identified with the three species Carex borealifujianica, C. hebecarpa, and C. ligulata (Fig 1). One way analysis of variance (ANOVA) was performed among these three groups. Leaf widths of Carex borealifujianica and C. hebecarpa were significantly narrower than that of C. ligulata (4.06±0.12 mm and 4.11±0.30 mm vs. 8.30±0.21 mm, respectively). Utricle densities of C. borealifujianica and C. hebecarpa were significantly sparser than that of C. ligulata (8.45 ±0.29 /cm and 6.52±0.36 /cm vs. 16.44±0.50 /cm, respectively). Utricle lengths of C. borealifujianica and C. ligulata were longer than that of C. hebecarpa (4.23±0.04 mm and 4.15±0.04 mm vs. 3.46±0.10 mm, respectively). In addition, pistillate scale lengths of C. borealifujianica and C. ligulata were longer than that of C. hebecarpa (2.49±0.04 mm and 2.49±0.04 mm vs. 1.77±0.03 mm, respectively).
Some characters of Carex borealifujianica distinguished it from both C. hebecarpa and C. ligulata, such as indumentum on basal sheaths (pilose vs. glabrous), number of staminate spikes (2 or 3 vs. solitary, respectively), and veins on utricles (conspicuous vs. inconspicuous). From the above-mentioned analysis, the diagnostic characters distinguishing the new species from Carex hebecarpa and C. ligulata are shown in Table 4.

PLOS ONE
Achene shape and surface sculpturing of C. borealifujianica and other six species in sect. Occlusae are shown in Fig 3. The trigonous achenes of most species were broadly obovoid, while those of C. maubertiana were obovoid. Moreover, only C. borealifujianica has achenes emarginate at the apex. The epidermal cells of all samples were irregularly 5-7-gonal, with straight inner anticlinal walls and a single silica body in each cell. Carex borealifujianica has achenes most similar to C. ligulata in micromorphology (Fig 3).

Phylogenetic relationships
The combined dataset contained 3057 bp aligned characters with the ETS, ITS, matK and trnL-F containing 616 bp, 628 bp, 776 bp and 1037 bp, respectively. The topologies of BI and ML trees were generally congruent and recovered a similar phylogeny to those of previous studies [11,12] (Fig 4). Six species in Carex sect. Occlusae clustered together and formed a distinct clade with high support (PP = 1, BS = 100%), which indicated this section is monophyletic. The three individuals of Carex borealifujianica formed a well-supported clade (PP = 1, BS = 100%). Carex ligulata is paraphyletic in a lineage that includes C. hebecarpa, indicating that further studies are required to clarify the relationship between these species.
This study indicated that Carex sect. Occlusae is monophyletic and recovered sister group to sect. Hallerianae (Aschers. & Graebn.) Rouy, which is consistent with previous studies [3,12]. The results can be more reliable than the findings of previous studies since the additional sampling of sect. Occlusae compared with only two samples previously.

Conclusions
Our study has found the population FJ-B from Fujian in SE China differs from Carex ligulata in several morphological characters and achene micromorphology. It was also found to display a distinct phylogenetic placement. Accordingly, we described it here as a new species. Haec species nova C. ligulatae Nees affinis est, sed foliis 2.5-5 mm latis, vaginis foliis sparse pilosis, spicis staminatis 2 vel 3, anguste clavatis, acheniis apice emarginatis differt.
Etymology. The specific epithet 'borealifujianica' refers to the type locality of this new species, Mount Wuyi, located in North Fujian Province.
Phenology. Flowering and fruiting from early May to mid-June. Distribution and habitat. Carex borealifujianica is known only from the type locality ( Fig  6). It grows on slopes, under forests or by streams at 220-320 m a.s.l.
Additional Conservation status: Vulnerable, VU B2ab(iii) (IUCN 2012). The occupancy of Carex borealifujianica is known to be less than 2000 km 2 , and it is distributed in no more than 10 locations. The quality of the habitat is continuously declining due to the disturbance caused by local tea plantation and tourism [53].
Key to the species in Carex sect. Occlusae from China (mainly modified from Flora of China [14]     Supporting information S1 Data. The original data of the observed and measured morphological characters for the principal component analysis.